Abstract
Enzymes accelerate the rate of chemical transformations by reducing the activation barriers of uncatalyzed reactions. For signaling enzymes, substrate recognition, binding, and product release are often rate-determining steps in which enthalpy-entropy compensation plays a crucial role. While the nature of enthalpic interactions can be inferred from structural data, the molecular origin and role of entropy in enzyme catalysis remains poorly understood. Using thermocalorimetry, NMR, and MD simulations, we studied the conformational landscape of the catalytic subunit of cAMP-dependent protein kinase A, a ubiquitous phosphoryl transferase involved in a myriad of cellular processes. Along the enzymatic cycle, the kinase exhibits positive and negative cooperativity for substrate and nucleotide binding and product release. We found that globally coordinated changes of conformational entropy activated by ligand binding, together with synchronous and asynchronous breathing motions of the enzyme, underlie allosteric cooperativity along the kinase’s cycle.
Highlights
Enzymes accelerate the rate of chemical transformations by reducing the activation barriers of uncatalyzed reactions
Using methyl-TROSY relaxation dispersion (RD) measurements, we discovered that synchronous breathing motions of the enzyme in the micro-tomillisecond time scale underscore positive binding cooperativity between Adenosine 5′-triphosphate (ATP) and substrate; while asynchronous dynamics characterize negative cooperativity between Adenosine 5′-diphosphate (ADP) and phosphorylated product
The thermodynamics of ATP and substrate binding to PKA-C has been extensively studied and it has been shown that nucleotide and the pseudo-substrate protein kinase inhibitor (PKI) peptide display approximately 400-fold binding cooperativity[21]
Summary
Enzymes accelerate the rate of chemical transformations by reducing the activation barriers of uncatalyzed reactions. While NMR-derived nanosecond–millisecond motions are likely not to be involved in the chemical step of catalysis[7], there is strong evidence that ligand binding affinities and kinetics of structural transitions are directly modulated by dynamics in the picosecond-to-nanosecond and micro-tomillisecond time scale, respectively[8,9,10,11,12] It remains unclear whether structural fluctuations during enzymatic catalysis are randomly distributed or are concerted to maximize catalytic efficiency. Using methyl-TROSY relaxation dispersion (RD) measurements, we discovered that synchronous breathing motions of the enzyme in the micro-tomillisecond time scale underscore positive binding cooperativity between ATP and substrate; while asynchronous dynamics characterize negative cooperativity between ADP and phosphorylated product. Our findings reveal that globally correlated motions along the kinase enzymatic cycle drive allosteric cooperativity and efficient turnover
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